It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or other-wise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et. al., Gene 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The complementarity of oligonucleotides has been used for inhibition of a number of cellular targets. Such complementary oligonucleotides are commonly described as being antisense oligonucleotides. Various reviews describing the results of these studies have been published including Progress In Antisense Oligonucleotide Therapeutics, Crooke, S. T. and Bennett, C. F., Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-129. These oligonucleotides have proven to be very powerful research tools and diagnostic agents. Further, certain oligonucleotides that have been shown to be efficacious are currently in human clinical trials.
It is well known, however, that oligonucleotides and their phosphorothioate analogues are of limited stability in blood and tissues. Also, since such compounds are being negatively charged they lack the ability to efficiently permeate biological membranes. Thus, both their oral bioavailability and cellular uptake are usually low. To overcome this problem, several types of modified oligonucleotides have been introduced. Among such oligonucleotides, backbone modified neutral oligonucleotides (namely, methylphosphonates and phosphate triester analogs) have gained a wide recognition. Recently, the latter modification has been even further developed with the introduction of bioreversible phosphate protecting groups into synthetic oligonucleotides. (see, e.g., Tosquellas, et al., Bioorg. Med. Chem. Lett. 1997, 7, 263; Mignet, et al., Bioorg. Med. Chem. Lett. 1997, 7, 851; Iyer, et al., Bioorg. Med. Chem. Lett. 1997, 7, 871; Iyer, et al., Tetrahedron 1997, 53, 2731). These groups are cleaved in a two-step process where the first step is either enzyme- or base-catalyzed hydrolysis of a (thio)ester group. The intermediate thus formed is unstable and spontaneously decomposes in neutral or slightly basic media to restore parent phosphorothioate diester and release either episulfide or quinonemethyde as a byproduct.
The synthesis of oligonucleotides with bioreversible phosphate protecting groups is complicated by the fact that one must deprotect phosphate and nucleic base moieties (typically by treatment with a base) while keeping the base labile (thio)ester function of the prodrug moiety intact. To a limited extent, this problem has been addressed with the (4-acyloxyphenyl)methyl group by finding more stable acyl residues and applying milder deprotection conditions. (see, Iyer, et al., supra). No selective deprotection method has yet to be reported, however, for the highly base-labile S-acyl 2-mercaptoethyl (SATE) group. Indeed, the reported synthesis for oligonucleotides having both phosphodiester and SATE phosphotriester units have involved postsynthetic alkylation of phosphorothioate oligonucleot Accordingly, there still remains a need for synthetic techniques and intermediates for such oligonucleotides, particularly techniques that do not involve postsynthetic modifications.